Methods for detecting dihydrotestosterone by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of dihydrotestosterone (DHT) in a sample using mass spectrometry. The methods generally involve ionizing DHT in a sample and detecting and quantifying the amount of the ion to determine the amount of DHT in the sample.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. application Ser. No. 12/272,663filed Nov. 17, 2008, and U.S. Application Ser. No. 61/103,202 filed Oct.6, 2008, which are incorporated by reference herein in their entiretyincluding all figures and tables.

FIELD OF THE INVENTION

The invention relates to the detection of dihydrotestosterone (DHT). Ina particular aspect, the invention relates to methods for detectingdihydrotestosterone (DHT) by mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Dihydrotestosterone (DHT) [(17β-hydroxy-5a-androstan-3-one)] is asteroid hormone with a molecular weight of 299.4 Daltons. DHT is apotent androgen synthesized by peripheral tissues from testosterone.Excessive DHT secretion can produce acne, hirsutism and virilization viaconversion to testosterone. DHT is a causal agent in prostatehyperplasia and measurements in blood can be used to assess complianceand response to inhibitors of testosterone to DHT conversion.

Mass spectrometric methods for measuring DHT in a sample have beenreported. See, e.g., Chang, Y., et al., Analyst 2003, 128:363-8; Caruso,D., et al., Neurochem Int 2008, 52:560-8; Wang, C., et al., Steroids2008, 73:1345-52; Zhao, M., et al., Steroids 2004, 69:721-6; Janzen, N.,et al., J Chroma B 2008, 861:117-22; Licea-Perez, H., et al., Steroids2008, 73:601-10; Kashiwagi, B., et al., J Andrology 2005, 26:586-91;Kashiwagi, B., et al., Urology 2005, 66:218-23; Umera, M., et al.,Cancer Sci 2007, 99:81-86; and Mohler, et al., U.S. patent applicationSer. No. 11/973,127 (filed Oct. 8, 2007).

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount ofdihydrotestosterone (DHT) in a sample by mass spectrometry, includingtandem mass spectrometry. Preferably, the methods of the invention donot include derivatizing DHT in a sample prior to the mass spectrometryanalysis.

In one aspect, methods are provided for determining the amount ofunderivatized dihydrotestosterone (DHT) in a body fluid sample. Methodsof this aspect include: (a) purifying DHT in a body fluid sample bysolid phase extraction; (b) ionizing DHT from the body fluid sample toproduce one or more DHT ions detectable by mass spectrometry, whereinthe produced ions are selected from the group consisting of a DHTprecursor ion with a mass to charge ratio of 291.10±0.50, and one ormore DHT fragment ions selected from the group consisting of 255.20±0.50and 79.20±0.50; and (c) detecting the amount of one or more DHT ions bymass spectrometry. Once the amount of the one or more DHT ions ismeasured, the amount of DHT ion(s) is used to calculate the amount ofunderivatized DHT in the test sample. In some embodiments, the massspectrometry is tandem mass spectrometry. In some embodiments, solidphase extraction and mass spectrometric analysis are conducted in anon-line fashion. In some embodiments, solid phase extraction isconducted as high turbulence liquid chromatography (HTLC). In someembodiments, the methods further comprising purifying DHT in a bodyfluid sample prior to mass spectrometry with high performance liquidchromatography (HPLC); preferably with on-line processing. In someembodiments, the body fluid sample is plasma or serum. In someembodiments, the methods have a limit of quantitation within the rangeof 5 ng/dL to 200 ng/dL, inclusive. In some embodiments, the amount ofone or more DHT ion(s) detected by mass spectrometry is used tocalculate the amount of underivatized DHT in a test sample by comparisonto an internal standard; preferably 16,16,17-d₃ dihydrotestosterone. Thefeatures of the embodiments listed above may be combined withoutlimitation for use in methods of the present invention.

In a second aspect, methods are provided for determining the amount ofunderivatized dihydrotestosterone (DHT) in a test sample by massspectrometry. Methods of this aspect include: (a) purifying DHT in atest sample with high turbulence liquid chromatography (HTLC); (b)ionizing DHT from a test sample to produce one or more DHT ionsdetectable by mass spectrometry; and (c) detecting the amount of one ormore DHT ions by mass spectrometry. In these methods, the amount of theDHT ion(s) measured is used to calculate the amount of DHT in the testsample. In some embodiments, the mass spectrometry is tandem massspectrometry. In some embodiments, purifying DHT in a test samplecomprises purifying with high performance liquid chromatography (HPLC);preferably configured for on-line processing. In some embodiments, thetest sample is a body fluid sample; preferably plasma or serum. In someembodiments, the DHT ions detectable by mass spectrometry include one ormore ions selected from the group consisting of ions with a mass/chargeratio of 291.10±0.50, 255.20±0.50, and 79.20±0.50. In some embodiments,the step of ionizing DHT includes generating a precursor ion with amass/charge ratio of 291.10±0.50, and generating one or more fragmentions selected from the group consisting of ions with a mass/charge ratioof 255.20±0.50 and 79.20±0.50. In some embodiments, the methods have alimit of quantitation within the range of 5.0 ng/dL to 200 ng/dL,inclusive. In some embodiments, the amount of one or more DHT ion(s)detected by mass spectrometry is used to determine the amount ofunderivatized DHT in a test sample by comparison to an internalstandard; preferably 16,16,17-d₃ dihydrotestosterone. The features ofthe embodiments listed above may be combined without limitation for usein methods of the present invention.

In at third aspect, methods are provided for determining the amount ofunderivatized dihydrotestosterone (DHT) in a body fluid sample by tandemmass spectrometry. Methods of this aspect include: (a) purifying DHTfrom a body fluid sample by high turbulence liquid chromatography(HTLC); (b) generating a precursor ion of said DHT having a mass/chargeratio of 291.10±0.50; (c) generating one or more fragment ions of aprecursor ion, wherein at least one of said one or more fragment ionscomprise a fragment ion selected from the group of fragment ions havinga mass/charge ratio of 255.20±0.50 and 79.20±0.50; and (d) detecting theamount of one or more of said ions generated in step (b) or (c) or both.The amount of ions detected is used to calculate the amount ofunderivatized DHT in a body fluid sample. In some embodiments, purifyingDHT from a test sample further comprises high performance liquidchromatography (HPLC); preferably configured for on-line processing. Insome embodiments, the body fluid sample is plasma or serum. In someembodiments, the methods have a limit of quantitation within the rangeof 5.0 ng/dL to 200 ng/dL, inclusive. In some embodiments, the amount ofone or more DHT ion(s) detected by mass spectrometry is used tocalculate the amount of underivatized DHT in the test sample bycomparison to an internal standard; preferably 16,16,17-d₃dihydrotestosterone. The features of the embodiments listed above may becombined without limitation for use in methods of the present invention.

Methods of the present invention involve the combination of liquidchromatography with mass spectrometry. In preferred embodiments, theliquid chromatography is HPLC. One preferred embodiment utilizes HPLCalone or in combination with one or more purification methods such asfor example HTLC and/or protein precipitation and filtration, to purifyDHT in samples. In another preferred embodiment, the mass spectrometryis tandem mass spectrometry (MS/MS).

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain preferred embodiments, DHT is measuredusing APCI in positive ion mode.

In preferred embodiments, DHT ions detectable in a mass spectrometer areselected from the group consisting of positive ions with a mass/chargeratio (m/z) of 291.10±0.50, 255.20±0.50, and 79.20±0.50. In particularlypreferred embodiments, a DHT precursor ion has m/z of 291.10±0.50, andone or more fragment ions are selected from the group consisting of ionshaving m/z of 255.20±0.50 and 79.20±0.50.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the endogenousDHT and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry.

A preferred internal standard is 16,16,17-d₃ dihydrotestosterone(16,16,17-d₃ DHT). In preferred embodiments, the internal standard ionsdetectable in a mass spectrometer are selected from the group consistingof positive ions with m/z of 294.10±0.50 and 258.20±0.50. Inparticularly preferred embodiments, an internal standard precursor ionhas m/z of 294.10±0.50; and an internal standard fragment ion has m/z of258.20±0.50.

In preferred embodiments, the presence or amount of the DHT ion isrelated to the presence or amount of DHT in a test sample by comparisonto a reference such as 16,16,17-d₃ dihydrotestosterone.

In certain preferred embodiments, the limit of quantitation (LOQ) of DHTis within the range of 5.0 ng/dL to 200 ng/dL, inclusive; preferablywithin the range of 5.0 ng/dL to 100 ng/dL, inclusive; preferably withinthe range of 5.0 ng/dL to 50 ng/dL, inclusive; preferably within therange of 5.0 ng/dL to 25 ng/dL, inclusive; preferably within the rangeof 5.0 ng/dL to 15 ng/dL, inclusive; preferably within the range of 5.0ng/dL to 10 ng/dL, inclusive; preferably about 5.0 ng/dL.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Derivatizing a molecule of an androgen, such as a moleculeof DHT, may be carried out with numerous derivatization reagents wellknown in the art. See, for example, Kashiwagi, B., et al., J Andrology2005, 26:586-91, and Kashiwagi, B., et al., Urology 2005, 66:218-23,which reports derivatization of DHT withfluoro-1-methylpyridinium-P-tolulene sulfonate prior to extraction. Asused herein, “underivatized” means not derivatized. Thus,dihydrotestosterone (DHT), without indication of derivatization, isunderivatized DHT.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedDHT parent or daughter ions by mass spectrometry. Relative reduction asthis term is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “test sample” refers to any sample that maycontain DHT. As used herein, the term “body fluid” means any fluid thatcan be isolated from the body of an individual. For example, “bodyfluid” may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column.

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about 4μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of ionization where the mechanism for the ionizationof molecule M is photon absorption and electron ejection to form themolecular ion M+. Because the photon energy typically is just above theionization potential, the molecular ion is less susceptible todissociation. In many cases it may be possible to analyze sampleswithout the need for chromatography, thus saving significant time andexpense. In the presence of water vapor or protic solvents, themolecular ion can extract H+ to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser diode thermal desorption is a technique wherein a samplecontaining the analyte is thermally desorbed into the gas phase by alaser pulse. The laser hits the back of a specially made 96-well platewith a metal base. The laser pulse heats the base and the heats causesthe sample to transfer into the gas phase. The gas phase sample is thendrawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of 20% and an accuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as two times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of DHT in a body fluid sample refersgenerally to an absolute value reflecting the mass of DHT detectable involume of body fluid. However, an amount also contemplates a relativeamount in comparison to another DHT amount. For example, an amount ofDHT in a body fluid can be an amount which is greater than a control ornormal level of DHT normally present.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of the coefficient of variation of assays of a blankand five standards used to determine the limit of quantitation of theDHT assay. Details are discussed in Example 5.

FIG. 2 shows the linearity of the quantitation of DHT in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 6.

FIGS. 3A and 3B show the correlation of DHT determination by anexemplary HPLC-MS method of the present invention with DHT determinationby a reference radioimmunoassay (RIA) method. The correlation shown inFIG. 3A was determined by linear regression. The correlation shown inFIG. 3B was determined by Deming analysis. Details are described inExample 10.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the present invention are described for measuring the amountof DHT in a sample. More specifically, mass spectrometric methods aredescribed for detecting and quantifying DHT in a test sample. Themethods may utilize high turbulence liquid chromatography (HTLC), toperform a purification of selected analytes, and combine thispurification with methods of mass spectrometry (MS), thereby providing ahigh-throughput assay system for detecting and quantifying DHT in a testsample. The preferred embodiments are particularly well suited forapplication in large clinical laboratories for automated DHT assay.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Particularly preferred samples includebodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid,or tissue samples. Such samples may be obtained, for example, from apatient; that is, a living person, male or female, presenting oneself ina clinical setting for diagnosis, prognosis, or treatment of a diseaseor condition. The test sample is preferably obtained from a patient, forexample, blood serum or plasma. A sample volume of about 0.5 mL ispreferred; however, samples of about 0.1 mL can be analyzed.

The present invention contemplates kits for an DHT quantitation assay. Akit for an DHT quantitation assay of the present invention may include akit comprising an internal standard, in amounts sufficient for at leastone assay. Typically, the kits will also include instructions recordedin a tangible form (e.g., contained on paper or an electronic medium)for using the packaged reagents for use in a measurement assay fordetermining the amount of DHT.

Calibration and QC pools for use in embodiments of the present inventioncan be prepared using “stripped” plasma or serum (stripped of DHT): forexample, double charcoal-stripped and delipidized serum. All sources ofhuman or non-human stripped plasma or serum should be checked to ensurethat they do not contain measurable amounts of DHT.

Sample Preparation for Mass Spectrometry

Test samples may be stored below room temperature. Test samples(including controls) stored below room temperature are first allowed tocome to room temperature and mixed by mechanical vortex. Internalstandard may be added to the test samples at this point.

The samples may then be prepared for mass spectrometry by liquid-liquidor solid-phase extraction. Various methods may be used to enrich DHTrelative to other components in the sample (e.g. protein) prior massspectrometry, including for example, liquid chromatography, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, extraction methods including ethyl acetateor methanol extraction, and the use of chaotropic agents or anycombination of the above or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Suchprotein purification methods are well known in the art, for example,Polson et al., Journal of Chromatography B 2003, 785:263-275, describesprotein precipitation techniques suitable for use in methods of thepresent invention. Protein precipitation may be used to remove most ofthe protein from the sample leaving DHT in the supernatant. The samplesmay be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, the use of protein precipitation such as forexample, formic acid protein precipitation, may obviate the need forHTLC or other on-line extraction prior to mass spectrometry or HPLC andmass spectrometry.

Accordingly, in some embodiments, protein precipitation, alone or incombination with one or more purification methods, may be used forpurification of DHT prior to mass spectrometry. In these embodiments,the methods may involve (1) performing a protein precipitation of thesample of interest; and (2) loading the supernatant directly onto theLC-mass spectrometer without using on-line extraction or HTLC.Alternatively, the methods may involve (1) performing a proteinprecipitation of the sample of interest; and (2) loading the supernatantonto a HTLC column to perform on-line extraction for furtherpurification prior to mass spectrometry.

One means of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select HPLC instruments and columns that are suitablefor use with DHT. The chromatographic column typically includes a medium(i.e., a packing material) to facilitate separation of chemical moieties(i.e., fractionation). The medium may include minute particles. Theparticles include a bonded surface that interacts with the variouschemical moieties to facilitate separation of the chemical moieties. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded or a cyano bonded surface. Alkyl bonded surfaces may include C-4,C-8, C-12, or C-18 bonded alkyl groups. In preferred embodiments, thecolumn is a cyano column. The chromatographic column includes an inletport for receiving a sample directly or indirectly from a solid-phaseextraction or HTLC column and an outlet port for discharging an effluentthat includes the fractionated sample.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a hydrophobic columnchromatographic system. In certain preferred embodiments, a cyanoanalytical column (e.g., a BetaBasic Cyano analytical column from ThermoScientific, Inc. (5 μm particle size, 50×2.1 mm), or equivalent) isused. In certain preferred embodiments, HTLC and/or HPLC are performedusing HPLC Grade 0.1% aqueous formic acid and 100% methanol as themobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, HTLC may be used for purification of DHT prior tomass spectrometry. In such embodiments, samples may be extracted usingan HTLC extraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column or onto an analytical HPLCcolumn prior to ionization. For example, sample extraction with an HTLCextraction cartridge may be accomplished with a large particle size (50μm) packed column. Sample eluted off of this column may then betransferred to an HPLC analytical column, such as a cyano analyticalcolumn, for further purification prior to mass spectrometry. Because thesteps involved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, DHT present in a test sample may be ionized byany method known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

DHT may be ionized in positive or negative mode. In preferredembodiments, DHT is ionized by APCI in positive mode. In relatedpreferred embodiments, DHT ions are in a gaseous state and the inertcollision gas is argon or nitrogen; preferably argon.

In mass spectrometry techniques generally, after the sample has beenionized, the positively charged or negatively charged ions therebycreated may be analyzed to determine a mass-to-charge ratio. Suitableanalyzers for determining mass-to-charge ratios include quadrupoleanalyzers, ion traps analyzers, and time-of-flight analyzers. The ionsmay be detected using several detection modes. For example, selectedions may be detected, i.e. using a selective ion monitoring mode (SIM),or alternatively, ions may be detected using a scanning mode, e.g.,multiple reaction monitoring (MRM) or selected reaction monitoring(SRM). Preferably, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and the mass/charge ratio.The voltage and amplitude may be selected so that only ions having aparticular mass/charge ratio travel the length of the quadrupole, whileall other ions are deflected. Thus, quadrupole instruments may act asboth a “mass filter” and as a “mass detector” for the ions injected intothe instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, an internalstandard is used to generate a standard curve for calculating thequantity of DHT. Methods of generating and using such standard curvesare well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, an isotopicallylabeled steroid may be used as an internal standard; in certainpreferred embodiments the standard is 16,16,17-d₃ dihydrotestosterone(16,16,17-d₃ DHT). Numerous other methods for relating the amount of anion to the amount of the original molecule will be well known to thoseof ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, DHT is detected and/or quantifiedusing MS/MS as follows. The samples are subjected to liquidchromatography, preferably HTLC; the flow of liquid solvent from thechromatographic column enters the heated nebulizer interface of an MS/MSanalyzer; and the solvent/analyte mixture is converted to vapor in theheated tubing of the interface. The analyte (e.g., DHT), contained inthe nebulized solvent, is ionized by the corona discharge needle of theinterface, which applies a large voltage to the nebulizedsolvent/analyte mixture. The ions, e.g. precursor ions, pass through theorifice of the instrument and enter the first quadrupole. Quadrupoles 1and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e.,selection of “precursor” and “fragment” ions in Q1 and Q3, respectively)based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is thecollision cell, where ions are fragmented. The first quadrupole of themass spectrometer (Q1) selects for molecules with the mass to chargeratios of DHT. Precursor ions with the correct mass/charge ratios areallowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. This process is calledcollision activated dissociation (CAD). The fragment ions generated arepassed into quadrupole 3 (Q3), where the fragment ions of DHT areselected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of DHT that may be usedfor selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of DHT. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, e.g., DHT, using calibration standardcurves based on peaks of one or more ions of an internal molecularstandard, such as 16,16,17-d₃ dihydrotestosterone.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Serum Sample and Reagent Preparation

Plasma samples were prepared by collecting blood in a Vacutainer tubewith no additives and allowed to clot for about 30 minutes at roomtemperature. Samples were then centrifuged and the serum separated fromthe cells. Samples that exhibited gross hemolysis were excluded.

Three stock solutions were prepared with DHT (Sigma Chemical Company,Cat. No. A7755, or equivalent). A DHT stock standard solution of 1 mg/mLin methanol was prepared in a volumetric flask. A portion of the DHTstock standard solution was then diluted by 1:100 to prepare a DHTintermediate stock standard solution of 1,000,000 ng/dL in methanol. Aportion of the intermediate stock standard solution was used to preparea second intermediate stock standard solution of 2,000 ng/dL inmethanol. The second intermediate stock standard solution was used toprepare a DHT working standard of 200 ng/dL in stripped serum.

16,16,17-d₃ dihydrotestosterone (CDN, Cat. No. D-5079, or equivalent)was used to prepare a 1.0 mg/mL in deuterated methanol 16,16,17-d₃dihydrotestosterone internal standard stock solution, which was used toprepare a 1,000,000 ng/dL in deuterated methanol 16,16,17-d₃dihydrotestosterone intermediate internal standard stock solution. 1.0mL of this intermediate stock solution was used to prepare a secondintermediate internal standard stock solution at 1000 ng/dL 16,16,17-d₃dihydrotestosterone in water. A 500 ng/dL 16,16,17-d₃dihydrotestosterone internal standard working solution was prepared bydiluting 20 mL of the second intermediate internal standard stocksolution with. DI water to volume in a 200 mL volumetric flask.

Example 2 Extraction of DHT from Samples Using Liquid Chromatography

Room temperature standards, controls, and patient samples were preparedfor liquid chromatography (LC) by first mixing by mechanical vortex.

300 μL of each vortexed standard, control, and patient sample was thentransferred to a well of a 96-well plate. 300 μL of 20% formic acid and100 μL of 500 ng/mL 16,16,17-d₃ dihydrotestosterone internal standardworking solution were then added to each. The plates were then vortexedand incubated at room temperature for 30 minutes before being loadedinto an autosampler drawer.

Sample injection was performed with a Cohesive Technologies Aria TLX-1HTLC system using Aria OS V 1.5 or newer software. An autosampler washsolution was prepared using 60% acetonitrile, 30% isopropanol, and 10%acetone (v/v).

The HTLC system automatically injected 100 μL of the above preparedsamples into a TurboFlow column (50×1.0 mm, 50 μm C-18 column fromCohesive Technologies) packed with large particles. The samples wereloaded at a high flow rate (5.0 mL/min, loading reagent 0.1% formicacid) to create turbulence inside the extraction column. This turbulenceensured optimized binding of DHT to the large particles in the columnand the passage of residual protein and debris to waste.

Following loading, the flow direction was reversed and the sample elutedoff to the analytical column (Thermo Scientific, BetaBasic Cyano column,5 μm particle size, 50×2.1 mm). A binary HPLC gradient was applied tothe analytical column, to separate DHT from other analytes contained inthe sample. Mobile phase A was 0.1% formic acid and mobile phase B was100% methanol. The HPLC gradient started with a 3% organic gradientwhich ramped to 50% in approximately 4.75 minutes. The separated samplewas then subjected to MS/MS for quantitation of DHT.

The specificity of the DHT against similar analytes was determined forthe following compounds (each at a concentration of 1000 ng/dL instripped serum): testosterone, estriol, dehydroepiandrosterone (DHEA),estrone, pregnenolone, estradiol, androstenedione, 17-OH pregnenolone,corticosterone, and aldosterone. No significant interference for any ofthese compounds was observed.

Example 3 Detection and Quantitation of DHT by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs all fromThermoElectron were used in the Examples described herein: Quantum TuneMaster V 1.2 or newer, Xcalibur V 1.4 SR1 or newer, TSQ Quantum 1.4 ornewer, and LCQuan V 2.0 with SP1 or newer. Liquid solvent/analyteexiting the analytical column flowed to the heated nebulizer interfaceof a Thermo Finnigan MS/MS analyzer. The solvent/analyte mixture wasconverted to vapor in the heated tubing of the interface. Analytes inthe nebulized solvent were ionized by APCI.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of 291.10±0.50 m/z. Ions entering Quadrupole 2 (Q2)collided with argon gas to generate ion fragments, which were passed toquadrupole 3 (Q3) for further selection. Simultaneously, the sameprocess using isotope dilution mass spectrometry was carried out with aninternal standard. 16,16,17-d₃ dihydrotestosterone. The following masstransitions were used for detection and quantitation during validationon positive polarity.

TABLE 1 Mass Transitions for DHT (Positive Polarity) Analyte PrecursorIon (m/z) Production (m/z) DHT 291.10 255.20 and 79.2 16, 16, 17-d₃294.10 258.2 dihydrotestosterone (internal standard)

Example 4 Intra-Assay and Inter-Assay Precision and Accuracy

Three quality control (QC) pools were prepared from charcoal-strippedhuman serum (Golden West Biologicals, Temecula, Calif.) spiked with DHTto a concentration of 25, 75, and 150 ng/dL, to cover the presumptivereportable range of the assay.

Ten aliquots from each of the three QC pools were analyzed in a singleassay to determine the coefficient of variation (CV (%)) of a samplewithin an assay. The following values were determined:

TABLE 2 Intra-Assay Variation and Accuracy Level I Level I Level III (25ng/dL) (75 ng/dL) (150 ng/dL) Mean (ng/dL) 26.8 73.83 145.23 StandardDeviation (ng/dL) 3.98 3.02 4.58 CV (%) 14.8% 4.09% 3.16% Accuracy (%)107.2% 98.44% 96.82%

Ten aliquots from each of the three QC pools were assayed over ten daysto determine the coefficient of variation (CV (%)) between assays. Thefollowing values were determined:

TABLE 3 Inter-Assay Variation and Accuracy Level I Level I Level III (25ng/dL) (75 ng/dL) (150 ng/dL) Mean (ng/dL) 24.51 75.18 152.31 StandardDeviation (ng/dL) 2.47 3.32 5.81 CV (%) 10.06% 4.42% 3.82% Accuracy (%)98.04% 100.24% 101.54%

Example 5 Analytical Sensitivity: Limit of Detection (LOD) and Limit ofQuantitation (LOQ)

The LOQ is the point where measurements become quantitativelymeaningful. The analyte response at this LOQ is identifiable, discreteand reproducible with a precision of 20% and an accuracy of 80% to 120%.The LOQ was determined by assaying analyte-stripped serum specimensspiked with DHT concentrations of 1.25, 2.5, 5.0, 10.0, and 20.0 ng/dL(five replicates at each level) then determining the CV. The resultswere plotted (shown in FIG. 1) and the LOQ was determined from the curveto be 5.0 ng/dL.

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as two standarddeviations from the zero concentration. To determine the LOQ for the DHTassay, blank samples of charcoal-stripped serum were run in tenreplicates. The results of these assays were statistically analyzed witha mean value of 1.0 ng/dL, and a standard deviation of 0.5 ng/dL. Thus,the LOD for the DHT was 2.0 ng/dL.

Example 6 Assay Reportable Range and Linearity

To establish the linearity of DHT detection in the assay, one blankassigned as zero standard and five spiked serum standards atconcentrations ranging from 10 to 200 ng/dL were assayed. Thecorrelation value of the concentration range tested (0 to 200 ng/dL) wasgreater than 0.995. A graph showing the linearity of the standard curveup to 200 ng/dL is shown in FIG. 2.

Example 7 Matrix Specificity

Matrix specificity was evaluated by diluting patient serum samplestwo-fold and four-fold with the following matrices: analyte-strippedserum (charcoal stripped serum, Cat. No. SP1070, Golden WestBiologicals, Inc.), normal human defibrinated serum (Cat. No. 1101-00,Biocell Labs, Carson, Calif. 90746, or equivalent), and deionized (DI)water. Two serum samples were spiked with the following concentrationsof DHT: 92.4 ng/dL, 64.1 ng/dL. The spiked serums were then diluted 2×and 4× with the above Matrices and analyzed. The study indicated thatall three matrices can be used to dilute samples with analyte valuesabove the linear range. The results of this study are presented in Table4.

TABLE 4 Matrix Specificity of DHT Expected Stripped Biocell DilutionConcentration Serum Serum DI Water Factor (ng/dL) (ng/dL) (ng/dL)(ng/dL) Sample 1 — 92.4 2x 46.2 47.7 50.8 49 4x 21.1 24.1 21.1 22.5Sample 2 — 64.1 2x 32.05 37.3 34.6 34.8 4x 16.03 19.1 18.9 16.4

Example 8 Recovery Studies

Quality control (QC) samples were used for recovery studies. Low, mid,and high QC samples were spiked with DHT to a concentration of 112.5,137.5, and 175 ng/dL, respectively

A recovery study of these DHT spiked samples was performed (five assaysat each concentration). Absolute recovery was calculated by dividing theDHT concentration detected in the pooled samples by the expected DHTconcentration in samples. The mean recoveries were 89.01%, 90.15%, and94.93%, respectively. All recoveries were acceptable, i.e., within therange of 80% to 120%.

Example 9 Specimen Studies

Specimens were derived from sample collection tubes with no additives(for serum), serum separator tubes (SST), EDTA tubes, or sodium heparintubes (36 samples, 18 from males and 18 from females). Four male and onefemale EDTA samples, as well as one female heparin sample, were excludedas unsuitable for analysis because of being either grossly hemolytic orlipemic. The remainder were tested for the applicability of the instantmethods to various sample types. Data analysis revealed that there waslittle difference between DHT levels detected in the various sampletypes (see Tables 5 and 6).

TABLE 5 Sample Type Comparisons for DHT, Males Sample Type Mean DHT(ng/dL) Standard Deviation (ng/dL) Serum 34.1925 20.5794 SST 39.249217.1014 EDTA 37.4059 15.6782 Heparin 42.7082 15.0552

TABLE 6 Sample Type Comparisons for DHT, Females Sample Type Mean DHT(ng/dL) Standard Deviation (ng/dL) Serum 26.6482 14.6048 SST 24.399118.8417 EDTA 28.9525 12.8975 Heparin 23.0536 18.8463

Example 10 Comparison of HTLC-MS and RIA Studies

A comparison study was performed using patient samples covering thereportable range, assayed by the instant methods with a referenceradioimmunoassay (RIA) method. Correlation was determined by linearregression (shown in FIG. 3A) and Deming analysis (shown in FIG. 3B).The correlation coefficient for linear regression analysis was 0.88.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1.-29. (canceled)
 30. A method for determining the amount ofdihydrotestosterone (DHT) in a body fluid sample, said methodcomprising: a. ionizing DHT from the body fluid sample via atmosphericpressure chemical ionization (APCI) under conditions suitable to produceone or more ions detectable by mass spectrometry; and b. determining theamount of one or more ions by mass spectrometry, and using the amountdetermined to calculate the amount of DHT in the body fluid samplewherein said method is capable of determining the amount of DHT in saidsample at concentrations of about 5 ng/mL or greater.
 31. The method ofclaim 30, wherein said ions comprise one or more ions selected from thegroup consisting of ions with a mass/charge ratio of 291.10±0.50,255.20±0.50, and 79.20±0.50.
 32. The method of claim 30, wherein saidions comprise an ion with the m/z ratio of 79.20±0.50.
 33. The method ofclaim 30, wherein said mass spectrometry is tandem mass spectrometry.34. The method of claim 33, wherein said ionizing comprises generating aprecursor ion with a mass/charge ratio of 291.10±0.50 and generating oneor more fragment ions selected from the group consisting of ions with amass/charge ratio of 255.20±0.50 and 79.20±0.50.
 35. The method of claim34, wherein said one or more fragment ions comprise a fragment ion witha m/z ratio of 79.20±0.50.
 36. The method of claim 30, wherein DHT fromthe body fluid sample has been purified by solid phase extraction (SPE)prior to ionization.
 37. The method of claim 36, wherein said SPE andmass spectrometry are conducted in an on-line fashion.
 38. The method ofclaim 36, wherein said SPE is conducted as high turbulence liquidchromatography (HTLC).
 39. The method of claim 30, wherein DHT from thebody fluid sample has been purified by high performance liquidchromatography (HPLC) prior to ionization.
 40. The method of claim 39,wherein said SPE and said HPLC are connected for on-line processingprior to ionization.
 41. The method of claim 30, wherein said body fluidsample is plasma or serum.
 42. The method of claim 30, wherein saidmethod is capable of wherein said method is capable of determining theamount of DHT in said sample at concentrations within the range of about5.0 ng/dL to about 200 ng/dL, inclusive.
 43. A method for determiningthe amount of dihydrotestosterone (DHT) in a test sample by massspectrometry, said method comprising: a. ionizing DHT, purified from thetest sample by solid phase extraction (SPE), under conditions suitableto produce one or more ions detectable by mass spectrometry; b.determining the amount of one or more ions by tandem mass spectrometry,wherein said tandem mass spectrometry comprises fragmenting a precursorion to generate a fragment ion with a mass to charge ratio of79.20±0.50, and c. using the determined amount of one or more ions tocalculate the amount of DHT in the body fluid sample.
 44. The method ofclaim 43, wherein said ionizing is conducted via atmospheric pressurechemical ionization (APCI).
 45. The method of claim 43, wherein said SPEis high turbulence liquid chromatography (HTLC).
 46. The method of claim43, wherein said DHT is further purified from the test sample with highperformance liquid chromatography (HPLC) prior to ionization.
 47. Themethod of claim 46, wherein said HTLC and said HPLC are configured foron-line processing.
 48. The method of claim 43, wherein said test sampleis a body fluid sample.
 49. The method of claim 43, wherein said testsample is plasma or serum.
 50. The method of claim 43, wherein precursorion has a mass to charge ratio of 291.10±0.50.
 51. The method of claim43, wherein said one or more fragment ions further comprises an ion witha m/z ratio of 255.20±0.50.
 52. The method of claim 43, wherein saidmethod is capable of determining the amount of DHT in said sample atconcentrations within the range of 5.0 ng/dL to 200 ng/dL, inclusive.